Molecular Cloning and Nucleotide Sequence Determination of Gene Encoding Streptomyces Subtilisin Inhibitor (SSI)

Abstract

A gene for Streptomyces subtilisin inhibitor (SSI) from Streptomyces albogriseolus S-3253 was cloned into E. coli plasmid pBR322 using two oligodeoxyribonucleotides corresponding to Asp68 to Pro77 and Asn99 to G1y107 of the protein, respectively. The SSI gene was localized on a 1.8-kbp BglII/SalI fragment. The nucleotide sequence of this 1.8-kbp fragment was determined by the dideoxy sequencing method. The amino acid sequence of the mature SSI coding region derived from the nucleotide sequence determination corresponded exactly to that from protein sequencing analysis. The nucleotide sequence analysis showed the presence of a putative signal peptide comprising 31 amino acids preceding the mature SSI region. The major transcriptional start point was identified to be 60 nucleotides upstream from the putative initiation codon for translation by the primer extension method. The -45 to -25 region upstream from transcriptional start point was quite homologous to that of CTC promoter of Bacillus subtilis. The overall G+C content of this 1.8-kbp fragment was 72%. On the other hand, an extremely high G+C content (96%) was found at the third letter of codons in the SSI coding region.